Download SIMULTANEOUS DETERMINATION AND VALIDATION OF AMOLODIPINE AND METAPROLOL

Academic Sciences
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 4, Suppl 5, 2012
Research Article
SIMULTANEOUS DETERMINATION AND VALIDATION OF AMOLODIPINE AND METAPROLOL
IN PHARMACEUTICAL DOSAGE FORMS BY REVERSE PHASE HPLC METHOD
B.VENKATA KIRAN, BATTULA SREENIVASA RAO*
Department of Chemistry, GITAM Institute of Technology, GITAM University, Visakhapatnam 530045, Andhra Pradesh, India.
Email: [email protected]
Received: 12 Sep, 2012, Revised and Accepted: 12 Oct, 2012
ABSTRACT
A rapid, specific and accurate isocratic HPLC method was developed and validated for the assay of Amolodpine besylate and Metaprolol in
pharmaceutical dosage forms. The assay involved an isocratic – elution of Amolodpine besylate and Metaprolol in ODS- C18 column using mobile
phase composition consists of 70:30(v/v) of Methanol and 0.1M potassium di hydrogen ortho phosphate with 0.1% W/V 1-octane sulfonic acid with
pH adjusted to 3.0.The wavelength of detection is 210 nm.The method showed good linearity in the range of 5.0-50.0 µg/mL for Amolodipine and 50
to 500 µg/mL for Metaprolol. The runtime of the method is 10 mins. The proposed method can be used for routine quality control samples in
industry in bulk and in finished dosage forms. In present study, a rapid specific precise and validated HPLC method for the quantitative estimation
of anolodpine and metaprolol in pharmaceutical dosage forms has been reported. The developed method can be applied to directly and easily to the
analysis of the pharmaceutical tablet preparations. The percentage recoveries were near 100% for given methods. The method was completely
validated and proven to be rugged. The excipients did not interfere in the analysis. The results showed that this method can be used for rapid
determination of amolodipine and metaprolol in pharmaceutical tablet with precision, accuracy and specificity.
Keywords: Amolodipine and metaprolol HCL, Assay, Reverse phase, HPLC.
INTRODUCTION
Amlodipine1, 2(AB),
chemically, 2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-1, 4- dihydro- 6-methyl-3, 5-pyridinedicarboxylic
acid3-ethyl, 5-methyl ester, is an anti-hypertensive and an
antianginal agent in the form of the besylate salt, amlodipine
besylate. Amlodipine3 exists in "left- and right-handed" chiral forms
(more accurately called the R-(+)Fig-1A and the S-(-)- enantiomers)
Fig-1B. Only the (S)- form of this molecule is active against
hypertension. When a mixture of (R) and (S)-forms (called a racemic
mixture) is used, patients may experience adverse side-effects such
as headache, dizziness, abdominal pain, flushing, peripheral oedema
etc. Obviously, in cases where only one of the enantiomers is active,
it is preferable to use only the chirally pure form. (-) Amlodipine is a
widely prescribed calcium channel blocking (CCB) antihypertensive
agent. However, amlodipine is a racemate with an equal proportion
of two enantiomers “S” and “R”, thus patients receiving amlodipine
are in fact taking two different drugs.S-Amlodipine and RAmlodipine which do not have the same level of antagonistic effect
on the calcium channel receptor.The S-enantiomer of amlodipine is
active and the R-enantiomer is inactive in terms of calcium channel
blocking activity. S(-)Amlodipine has 1000 fold stronger calcium
channel blocking activity than R- amlodipine.S(-)Amlodipine is
therefore responsible for all of the CCB-mediated pharmacodynamic
action of amlodipine including its anti-anginal activity without the
concomitant liability of adverse effects associated with the racemic
mixture of amlodipine. As S-Amlodipine is more active than the Renantiomer, the faster release of S-enantiomer from a dosage form is
very essential to treat the emergency conditions like hypertension
and angina.Like other calcium channel blockers, amlodipine acts by
relaxing the smooth muscle in the arterial wall, decreasing
peripheral resistance and hence reducing blood pressure; in angina
it increases blood flow to the heart muscle. Amlodipine besylate is a
white crystalline powder with a molecular weight of 567.1. It is
slightly soluble in water and sparingly soluble in ethanol.
Amlodipine Besylate Tablets USP are formulated as white to offwhite tablets equivalent to 2.5 mg, 5 mg and 10 mg of Amlodipine
for oral administration. In addition to the active ingredient,
Amlodipine besylate, each tablet contains the following inactive
ingredients: dibasic calcium phosphate anhydrous, magnesium
stearate, microcrystalline cellulose and sodium starch glycolate. The
usual initial antihypertensive oral dose of Amlodipine besylate
tablet is 5 mg once daily with a maximum dose of 10 mg once daily.
Bioavailability is 64% to 90%. About 93% is protein
bound..Metoprolol Succinate Fig-2(RS)-1- (isopropylamino)-3-[4-(2-
methoxyethyl)phenoxy] propan-2-ol, is a cardio selective drug used
in the treatment of hypertension and various cardiovascular
disorders. The action of Metoprolol succinate4 is mediated through
the beta1-selectiveadrenoceptorblockage, thus causing reduction in
heart rate and cardiac output. It is a beta1-selective drug which
belongs to the chemical class of beta blockers and is(±)1(isopropylamino)-3-[p-(2-methoxyethyl)phenoxy]-2propanolsuccinate (2:1)(salt) with molecular formula of
(C15H25NO3)2. C4H6O4 and molecular weight of.652.81. At low doses,
metoprolol selectively blocks cardiac β-1-adrenergic receptors with
little activity against β2-adrenergic receptors of the lungs and
vascular smooth muscle. Receptor selectivity decreases with higher
doses. Unlike propranolol and pindolol, metoprolol does not exhibit
membrane-stabilizing or intrinsic sympathomimetic activity.
Membrane-stabilizing effects are only observed at doses much
higher than those needed for β-adrenergic blocking activity.
Metoprolol possesses a single chiral centre and is administered as a
racemic mixture.
The Adsorbtion of Metaprolol is rapid and complete. Absorbed
readily and completely for the GI tract (oral); peak plasma
concentrations after 1.5-2 hours.Distributed widely, crosses the
placenta and enters breast milk. Protein-binding: 12%.Its water
solubility is 16.9mg/ml.The melting point of Metaprolol in its
tartrate form is about 1200C.Metaprolol exist in salts forms of tatrate
and succinate. For healthy adults, the starting dose is 25 to 100 mg
daily in single or divided doses and the maximum dose is 400 to 450
mg/day Metoprolol tartrate was developed by Novartis and received
approval in the United States August 7, 1978.Generic metoprolol
succinate was developed by Sandoz and received approval in
the United States on July 31, 2006. It is marketed under the brand
name Lopressor by Novartis, and Toprol-XL (in the USA); Selokeen
(in the Netherlands); as Minax by Alphapharm (in Australia), Metrol
by Arrow Pharmaceuticals (in Australia), as Betaloc by AstraZeneca,
as Neobloc by Unipharm (in Israel) and as Corvitol by Berlin-Chemie
AG (in Germany). In India, this drug is available under the brand
names of Metolar and Starpress. A number of generic products are
available as well. The active substance metoprolol is employed
either as metoprolol succinate or metoprolol tartrate (whereas 100
mg metoprolol tartrate corresponds to 95 mg metoprolol succinate),
respectively as prolonged-release or conventional-release
formulation. Both Amodlopine besylate and Metaprolol is used as
combination for treatment of Hypertension, they are many generic
version supplied by various companies in various dosage forms such
as AMTAS-M (INTAS) containing 25 mg and 5 mg of Metaprolol and
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Amodlopdine respectively, CORVADIL-M(UNICHEM) containing
Metaprolol and Amolodpine 25 mg and 2.5 mg respectively,
SITELOL-AM(SANOFI-AVENTIS) Containing 25 mg and 5mg of
Metaprolol and Amolodipine respectively. METSTAMLO(DRL)
capsules Contaning 25 mg and 5mg of Metaprolol and Amolodpine
respectively.METALOL-AM of 25and 50 Tablets (CIPLA) containing
relatively corresponding amounts of both metaprolol and
Amolodpine respectively. From literature survey separate methods
have been developed for amolodipine and metaprolol. Several
methods have been reported for the quantitative determination of
amolodipine in bulk, and pharmaceutical and biological samples.
These methods include UV-Visible spectrophotometric and UVderivative 5-7, , HPTLC8-12, , HPLC-UV-detector12-19, HPLCAmeperometric detection20-21, HPLC with Mass-spectrophotometer
detector22,
Gas-chromotography-chromatography-Massspectrophtometry23. Similarly individual methods have been
developed
for
metaprolol
which
include
UV-Visible
spectrophotometric and UV-derivative24, HPLC-UV-detector25-34,
HPLC
with
solid-phase
HPLC-UV-Floroscence
detector35,
extraction36, Super-critical fluid liquid chromatography37,
Hyphenated techniques such as GC-MS38.
for amolodipine and 50, 100, 150, 200, 300, 400, and 500 µg/ml of
metaprolol from first standard stock solution of the linear
calibration standard auto sampler for analysis. Three quality control
samples were at the concentrations of 12.62, 25.24 and 37.86 µg/ml
for amolodipine and 126.88, 253.77 and 375.77 µg/ml of metaprolol
were prepared for analysis representing low, medium and high
concentration respectively.
However from literature surevey it has been observed very few and
scanty methods have been developed for determination of
Amolodpine for both (R) and (S)-enatiomeric39-40 forms and
simultaneous determination of amolodpine and metaprolol by
HPLC41-42Besides many of the above said methods could not separate
amolodpine in its R and S forms.Hence the author has attempted to
develop a method for simultaneous determination of metoprolol and
Amolodipine. By HPLC 43-44 and UV 45.
Method Validation
MATERIALS AND METHODS
Chemicals and reagents
Amolodipine (99.92%) working standard was gift samples from
corpuscle research solutions and Metaprolol (99.89% pure, was
procured from corpuscle research solutions. acetontrile (HPLC
grade) was obtained from Qualigens fine chemicals. Milli-Q water
was purchased from Ranbaxy fine chemicals limited (RFCL). 1Octane-sulfonic-Acid was purchased from Merck. All chemicals used
were of HPLC grade.
Sample preparation
Commercially available tablets of are taken from two different
brands and tested for assay. Twenty tablets of each brand are taken
and crushed to powder. A powder equivalent to 1mg/ml and
1mg/ml of amolodpine and metaprolol is taken and transferred into
a stoppered conical flask to which 25ml of methanol is added. The
contents are transferred into a stoppered flask and shaken for 20
mins to extract the drug. Contents are carefully transferred into a
centrifuge tube and centrifuged for 4000 rpm for 20 mins. The
supernatant liquid is taken and diluted with diluents, to obtain
approximately final concentration of 10 µg/ml and 100 µg/ml. This
sample is analyzed in triplicate. The accuracy and concentration is
determined using regression equation.
System Suitability
System Suitability is an integral part of the LC method. They are used
to verify that resolution. reproducibility are adequate. This is based on
the concept that equipment, electronics, analytical operations,
analytical operator, and sample constitute an integral system that can
be evaluated as such. The system suitability was assessed by six
replicate analysis of the drug at a concentration of 25 µg/ml and 250
µg/ml concentrations of amolodipine and metaprolol respectively.The
acceptance criterion is ± 1% for the per cent coefficient of the variation
for the peak area and retention times for the both drug and internal
standard. The parameters which we have evaluated are USP
Theoretical plates, USP tailing factor and USP Resolution. The
following formulas has been used for the calculation of
USP resolution: Rs = 2(T1-T2)/W1-W2
USP Theoretical plates = 5.54 [TR/WR]2
Instrumentation
USP Tailing = W0.5 / 2d
The HPLC system consisted of a Shimadzu Class VP Binary pump LC10Atvp, SIL-10Dvp Auto sampler, CTO-10Avp column temperature
Oven, PDA-UV Detector. All the components of the system are
controlled using SCL-10Avp System Controller. Data acquisitions
was done using LC-solution software. The mobile phase consists of
70:30 (v/v) of Methanol and 0.1M potassium dihydrogen
orthophosphate with 0.2% w/v 1-octane sulfphonic acid as modifier,
with pH adjusted to 3.0 with otho phosphoric acid. operated on
isocratic mode. Analysis was carried out at 210 nm. The
chromatographic separation of Amodlodipine and metaprolol was
carried out using Waters spherisorb, 250X 4.6 mm X 5µm, ODS-2
Column. The flow rate is 1.0 ml/min. The injection volume is
20µL.The run time of the method is 10 mins. Diluents consists of
mobile phase.
Detection and Quantization Limits (sensitivity)
Preparation of solutions
Drug stock solution and internal standard
Two different Stock solution of Amolodipine working standard and
Metaprolol was prepared by dissolving accurately weighed 10mg of
drug in 10 ml of methanol , so that final concentration is
1mg/1ml.The prepared stock solution is stored in 40C protected
from light. Suitable dilutions of both the drugs were prepared by
using diluent solution.
Calibration standards and quality control samples
An eight point linear calibration curve standards were prepared
using diluents solutions in the concentration range of 5.0 to
50.20µg/ml, and 50 to 500 µg/ml for amolodipine and Metaprolol
respectively. Calibration standards were prepared at the
concentration of 5.05, 10.09, 15.14, 20.19, 31.55, 44.17, 50.47 µg/ml
Limits of detection (LOD) Fig-3 and quantization (LOQ), Fig-4 were
estimated from both linearity calibration curve method and signal to
noise ratio method. The detection limit was defined as the lowest
concentration level resulting in a peak area of three times the
baseline noise. The quantization limit was defined as the lowest
concentration level that provided a peak area with signal to noise
ratio higher than 10, with precision (%CV) and accuracy with (±)
10%
Linearity (Calibration Curve)
The calibration curve were constructed with eight concentration
ranging from 2.01 to 50.20 µg/ml. The peak area ratio of the drug to
the internal standard was evaluated by linearity graph. The linearity
was evaluated by linear regression analysis, which was calculated by
least square method. It is depicted in Fig-5(a), Fig-5(b) and Fig5(c) respectively.
Accuracy and Precision
Accuracy of assay method was determined for both intra-day and
inter-day variations using triplicate analysis of the QC samples.
Precision of the assay was determined by repeatability (intra-day)
and intermediate precision (inter-day). Repeatability refers to the
use of the analytical procedure within the laboratory over the
shorter period of the time that was evaluated by assaying the QC
samples during the same day. Intermediate precision was assessed
by comparing the assays on different days (3 days).
Specificity
Specificity of the method was determined by injecting 3 samples
402
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
1)
Chromatogram of Blank.(Fig-6).
2)
Chromatogram of “R” and “S”-Amolodipine besylate.Fig-7).
3)
Chromotogram of Metaprolol.(Fig-8).
A less than 20% interference of the peak area at the retention time
of the drug in the blank sample and zero blank samples are taken as
acceptance criteria for the analyte. The interference of the internal
standard the peak area at the retention time of the internal standard
must the less than 5% in the blank sample. Specificity is also
observed in the degradation study of the drug. None of the degraded
products must interfere with the quantification of the drug.
Stability
The stability of the drug is determined by using QC samples for the
short term stability by keeping at room temperature upto 12 hours
and then analyzing them. Further, auto-sampler stability for upto 24
hrs was also established.
RESULTS AND DISCUSSIONS
Method Development and Validation
The HPLC procedure was optimized with a view to develop a
stability indicating assay method. Different permutations and
combinations, at different pH values ranging from pH 3.0 to pH 11.0
using various columns like Hypersil-BDS-C18, Symmetry C18, YMCPACK C18YMC-PACK PRO, have been tried with different buffer salts
such ammonium acetate, potassium-di-hydrogen orthophosphate,
di-potassium hydrogen orthophosphate, in combination with
acetonitrile, methanol and tetrahydrofuran. However good
resolution, less tailing and high theoretical plates are obtained with
Waters spherisorb column C18 250 X 4.6 mm, 5 µm The mobile
phase consists of 70:30 (v/v) of Methanol and 0.1M potassium
dihydrogen orthophosphate with 0.2% w/v 1-octane sulfphonic acid
as modifier, with pH adjusted to 3.0 with otho phosphoric acid.
operated on isocratic mode. Analysis was carried out at 210 nm. The
chromatographic separation of Amodlodipine and metaprolol was
carried out using Waters sphrosorb, 250X 4.6X 5µm, ODS-2 Column.
The flow rate is 1.0 ml/min.The injection volume is 20µL.The run
time of the method is 10 mins. Diluents consists of mobile phase.
The column temperature is maintained at 250 C. At the reported
flow rate peak shape was excellent, however increasing or
decreasing the flow rate increased the tailing factor and resulting in
poor peak shape and also resolution between the drug and internal
standard also decreased. Hence 1.0 ml/min was optimized flow rate
decreasing the consumption of the mobile phase, which in turn
proves to be cost effective for long term routine quality control
analysis. There was no interference in the drug and internal
standard, from the blank. The peak shape and symmetry were found
to be good when the mobile phase composition of 70:30 v/v was
used, with better resolution between the (R )- and (S )-Amolidipine
and metaprolol drugs.
concentration is = 0.50 µg/ml and 5.0 µg/ml for amolodipine and
metaprolol respectively.
Linearity
The calibration curve constructed was evaluated by its correlation
coefficient. The peak area ratio of the drugs, (S)-amolodipine, (R)amolodipine and metaprolol was linear, and the range, is 5.0 and
50.00µg/ml, for amolodipine and 50 to 500µg/ml, for
metaprolol.The linearity was determined in three sets, the
correlation coefficient (R2) was consistently greater then 0.990
(Table 4(a), Table 4(b), Table 4(c)). From the data in Table 4(a),
Table 4(b), Table 4(c) regression equation, limit of quantification
and limit of detection was determined from the calibration curve
method.
Regression equation:
(1) y = 33472x – 41210, for (R )-amolodipine.
(2) y = 78656x – 11305, for (S )-amolodpine.
(3) y = 24802x + 23877, for metaprolol.
Accuracy and Precision
Accuracy and precision calculated for the QC samples during the
intra- and inter –day run are given the (Table 5(a), Table 5(b),
Table 5(c)).The intra-day (day-1) and inter-day accuracy ranged
from 99.15 to 102.18. The results obtained from intermediate
precision (inter-day) also indicated a good method precision.All the
data were within the acceptance criteria.
Specificity
Specificity was determined from Blank (Fig-6) representative
chromatograms of (S)- amolodipine, (R)-amolodipine (Fig-7) and
representative chromatogram of metaprolol (Fig-8).
Stability
Stability studies were done for short term stability upto 12 hrs, auto
sampler stability upto 24hrs at three different concentrations of low
QC, medium QC, High QC levels conditions and the mobile phase is
stable upto 72 hrs.These data can be interpreted from Table 6(a)
and Table 6(b), for amolodpine and metaprolol respectively.
Robustness study
Robustness is the measure of method capacity to remain unaffected
by deliberate small changes in the chromatographic conditions. The
experimental conditions were deliberately altered to test evaluate
the robustness of the method. The impact of flow-rate(1.0±0.1),
column temperature (150C 250C, 300C)changes and effect of mobilephase composition(±10%) was evaluated on the important system
suitability factors such as retention time, theoretical plates, tailing
factor, and resolution were studied. The experimental results were
presented in the (Table 7(a) and Table 7(b) Table 7(c)).
Method Validation
Application of the method to dosage forms
System Suitability
The % RSD of the peak area and the retention time for both drug and
internal standard are within the acceptable the range Table
1(a).The efficiency of the column was expressed as the number of
USP theoretical plates for the six replicate injections was around
4946 ± 85 and the USP tailing factor was 1.41 ± 0.01 for (R )amolodipine, 6615 ± 22 and 1.57±0.01 and the USP resolution
between the (R )- and (S )-amolodipine is 17.28 ± 0.08. From Table
1(b) the efficiency is determined by USP theroretical plates which is
around 6498 ± 53, the USP tailing is around 1.85 ± 0.03, and USP
resolution between metaprolol and (S )-amolodipine is 8.64 ± 0.02.
The HPLC method developed is sensitive and specific for the
quantitative determination of Metaprolol and Amolodipine besylate.
Also the method is validated for different parameters, hence has
been applied for the estimation of drug in pharmaceutical dosage
forms. Metaprolol and Amolodipine besylate tablets of 25mg, 5mg
strength from two different manufacturers were evaluated for the
amount of Metaprolol and Amolodipine besylate tablets. The amount
of Metaprolol in tablet 1 is 97.06 ± 0.56 and tablet 2 is 99.24 ± 0.32
and amolodpine besylate in tablet 1 is 98.79 ± 0.86 and tablet 2 is
99.59 ± 0.90 (Table 8).None of the tablets ingredients interfere with
the analytic peak.
Detection and quantization limits (sensitivity)
CONCLUSION
(Fig-3) and (Fig-4) represents the six replicate injections of the limit
of detection and limit of quantification. The method is found to be
sensitive which can be determined from the data obtained from the
(Table 2) and (Table 3).The LOD Concentration is = 0.25µg/ml, and
2.5 µg/ml for amolodipine and metaprolol respectively. The LOQ
A rapid sensitive and specific method for the determination of
amolodipine and metaprolol in the pharmaceutical; formulations has
been developed using external and simultaneous determination
method. The method gave accurate and precise results in the
concentration range of 5.0-50.00µg/ml, for amolodipine and 50.00
403
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
to 500µg/ml for metaprolol respectively. 70:30(v/v) of Methanol
and 0.1M potassium di hydrogen ortho phosphate with 0.1% W/V 1octane sulfonic acid with pH adjusted to 3.0.The wavelength of
detection is 210 nm. The flow rate of 1.0 ml/min. The retention
times of (R) –amolodpine, (S )-amolodpine and metaprolol are 2.11
± 0.01, 8.51 ± 0.03 and 5.52 ± 0.01 respectively.
W1, W2 = peak width 1 and 2 at the base line.
Abbreviations
W0.5 = width of the peak at 1/20 of peak height.
Rs = USP Resolution.
d = distance between perpendicular dropped from peak maximum
to the leading edge of the peak at 1/20 of peak height.
T1, T1 = retention time of the Peak 1, Peak 2.
TR = retention time along the baseline from the point of injection to
the perpendicular dropped from the maximum of the peak
corresponding to the analyte.
WR = width of the peak at half the height.
Table 1(a): System Suitability Study
Inj-01
Inj-02
Inj-03
Inj-04
Inj-05
Inj-06
Mean
S.D
RSD
R-Amolodipine
R.T
T.P
2.08
4830
2.12
4932
2.12
5067
2.12
4927
2.12
5022
2.12
4899
2.11
4946.17
0.02
85.60
0.77
1.73
Tailing
1.4
1.43
1.41
1.41
1.4
1.41
1.41
0.011
0.78
S-Amolodipine
R.T
T.P
8.56
6594
8.54
6601
8.5
6606
8.49
6608
8.49
6632
8.48
6652
8.51
6615.5
0.032
22.02
0.38
0.33
Resolution
Tailing
1.56
1.57
1.58
1.59
1.57
1.58
1.575
0.010
0.67
17.4
17.29
17.29
17.20
17.26
17.21
17.28
0.072
0.42
Table 1(b): System Suitability Study
Inj-01
Inj-02
Inj-03
Inj-04
Inj-05
Inj-06
Mean
S.D
RSD
Metaprolol
R.T
5.52
5.54
5.52
5.51
5.51
5.51
5.52
0.012
0.21
Resolution
T.P
6394
6528
6495
6518
6513
6545
6498.83
53.95
0.83
Tailing
1.8
1.88
1.86
1.89
1.85
1.84
1.85
0.03
1.73
8.68
8.64
8.62
8.61
8.64
8.63
8.64
0.02
0.28
Table 2: Limit of detection
Injection. No
01
02
03
04
05
06
Mean
S.D
RSD
R-Amolodipine
26942
27190
27697
27456
27543
27987
27469.17
369.19
1.34
S-Amolodipine
18065
14264
16840
17786
17498
17543
16999.33
1400.44
8.24
Metaprolol
57142
51879
53392
54345
54564
54634
54326
1728.69
3.18
Table 3: Limit of Quantification
Injection. No
01
02
03
04
05
06
Mean
S.D
RSD
R-Amolodipine
111323
111228
111706
111675
111543
111198
111445.5
225.21
0.20
S-Amolodpine
37127
37625
37706
37435
37453
37564
37485
203.16
0.54
Metaprolol
111471
111424
112872
111675
111876
111956
111879
530.49
0.47
Table 4(a): Results and regression analysis of linearity data of R-Amolodipine
Mean ± S.D(n=3)
Slope
Intercept
Correlation coefficient(R2)
33472 ± 0.06
-41210± 0.08
0.999 ± 0.0003
Each mean value is a result of triplicate analysis (n=3)
404
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Table 4(b): Results and regression analysis of linearity data of S-Amolodipine
Mean ± S.D(n=3)
Slope
Intercept
Correlation coefficient(R2)
38656 ± 0.06
-11305± 0.08
0.998 ± 0.0003
Each mean value is a result of triplicate analysis (n=3)
Table 4(c): Results and regression analysis of linearity data of Metaprolol
Mean ± S.D(n=3)
Slope
Intercept
Correlation coefficient(R2)
24802 ± 0.06
23807 ± 0.08
0.994 ± 0.0003
Each mean value is a result of triplicate analysis (n=3)
Table 5(a): Intra-day and Inter-day precision and accuracy of HPLC assay of (R)-amolodipine
Day=1
Mean (n=3)
S.D
R.S.D
Recovery(%)
Day=2
Mean(n=3)
S.D
R.S.D
Recovery(%)
Day-3
Mean (n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
12.62 µg/ml
25.24 µg/ml
37.86 µg/ml
384538.3
459.5197
0.119499
100.7887
812357
1624.629
0.19999
101.0338
1226008
1818.633
0.148338
99.99751
386164
1025.655
0.265601
101.4066
814087
4566.34
0.560915
101.2386
1225262
3938.109
0.32141
99.15198
386003.7
1051.966
0.272527
101.1356
809787
5770.014
0.287129
100.7296
1223920
3368.271
0.275204
99.83269
Each mean value is a result of triplicate analysis (n=3)
Table 5(b): Intra-day and Inter-day precision and accuracy of HPLC assay of (S)-amolodipine
Day=1
Mean (n=3)
S.D
R.S.D
Recovery(%)
Day=2
Mean(n=3)
S.D
R.S.D
Recovery(%)
Day-3
Mean (n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
12.62 µg/ml
25.24 µg/ml
37.86 µg/ml
996594.7
2984.33
0.30
99.54
2009734
5669.38
0.28
100.80
3004266
1377.12
0.05
100.26
996155.3
2852.65
0.29
101.49
2009734
5669.38
0.28
101.61
3007316
7239.91
0.24
101.61
995972
1051.97
0.27
101.14
2009557
5770.01
0.29
100.73
3008385
9247.74
0.31
101.40
Each mean value is a result of triplicate analysis (n=3)
Table 5(c): Intra-day and Inter-day precision and accuracy of HPLC assay of Metaprolol
Day=1
Mean (n=3)
S.D
R.S.D
Recovery(%)
Day=2
Mean(n=3)
S.D
R.S.D
Recovery(%)
Day-3
Mean (n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
126.88 µg/ml
253.77 µg/ml
375.77 µg/ml
3238524
37659.81
1.16
100.15
6303843
2433.858
0.04
99.78
9418967
23158.41
0.25
100.81
3238473
37236.79
1.15
102.15
6303730
1836.932
0.03
99.78
9417750
11810.22
0.13
100.79
3239393
36155.95
1.12
102.18
6303307
2040.434
0.03
99.77
9406182
2305.39
0.02
100.67
Each mean value is a result of triplicate analysis (n=3)
405
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Table 6(a): Short-term, long term and auto-sampler stability of (R )-amolodpine
Short term stability
(12 hrs)
Mean (n=3)
S.D
R.S.D
Recovery(%)
Auto sampler stability(24 hrs)
Mean(n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
12.62 µg/ml
25.24 µg/ml
37.86 µg/ml
374210
2417.469
0.643164
98.34366
802441
5712.005
0.706325
100.6002
1217409
5183.077
0.423809
99.75814
372098
2236.3
0.596689
96.01184
800987
6786.191
0.840859
100.4062
1206075
9450.504
0.775426
99.42474
Table 6(b): Short-term, long term and auto-sampler stability of (S )-amolodpine
Short term stability
(12 hrs)
Mean (n=3)
S.D
R.S.D
Recovery(%)
Auto sampler stability(24 hrs)
Mean(n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
12.62 µg/ml
25.24 µg/ml
37.86 µg/ml
993456
2984.333
0.299453
101.2212
2006477
2593.831
0.129446
101.6373
3001202
3243.113
0.107998
101.1616
989856
4427.479
0.444901
100.8585
2000134
1573.54
0.078623
101.3178
3000089
3005.572
0.100112
101.1242
Table 6(c): Short-term, long term and auto-sampler stability of Metaprolol
Short term stability
(12 hrs)
Mean (n=3)
S.D
R.S.D
Recovery(%)
Auto sampler stability(24 hrs)
Mean(n=3)
S.D
R.S.D
Recovery(%)
Nominal concentration
126.88 µg/ml
253.77 µg/ml
375.77 µg/ml
3165439
19234.74
0.61
99.83
6302098
3321.786
0.05
99.75
9405057
23109.7
0.25
100.66
3160970
18335.36
0.58
99.69
6300089
4930.179
0.08
99.72
9390100
12430.46
0.13
100.50
Table 7(a): Effect of Various parameters in assessment of method for (R )-Amolodipine
Parameters
Variation
Flow rate
0.9ml/min
1.0ml/min
150C
250C
300C
100% organic
110% organic
Column temperature
Mobile phase
Analyst-Analyst Variation
Analyst-1
Analyst-2
Analyst-3
Analyst-4
Observed values
R.T
T.P
2.38
4946
2.11
4947
2.11
4804
2.11
4947
2.11
4869
2.11
4947
1.83
3992
Tailing
1.44
1.41
1.4
1.41
1.39
1.41
1.6
Resolution
18.16
17.28
17.64
17.28
17.63
17.28
13.6
2.11
2.12
2.11
2.07
1.41
1.47
1.48
1.44
17.28
17.66
17.64
17.71
4947
4778
4786
4766
406
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Table 7(b): Effect of Various parameters in assessment of method for (S )-Amolodipine
Amolodipine
Variation
Parameters
Flow rate
Column temperature
Mobile phase
0.9ml/min
1.0ml/min
150C
Observed values
R.T
T.P
6.33
6615
5.52
6616
5.68
6421
Tailing
1.94
1.85
1.85
Resolution
17.64
17.28
17.63
250C
300C
100% organic
110% organic
5.52
5.68
5.52
4.7
6616
6424
6616
6544
1.85
1.86
1.85
2.27
17.28
13.6
5.52
5.69
5.68
5.89
6616
6535
6475
6539
1.85
1.84
1.84
1.86
17.64
17.77
17.64
17.28
Analyst-Analyst Variation
Analyst-1
Analyst-2
Analyst-3
Analyst-4
17.28
Table 7(c): Effect of Various parameters in assessment of method for Metaprolol
Parameters
Variation
Flow rate
0.9ml/min
1.0ml/min
150C
250C
300C
100% organic
110% organic
Column temperature
Mobile phase
Analyst-Analyst Variation
Analyst-1
Analyst-2
Analyst-3
Analyst-4
Observed values
R.T
T.P
9.98
6920
8.51
6615
8.94
6589
8.51
6615
8.94
6620
8.51
6615
7.84
6850
Tailing
1.65
1.58
1.54
1.58
1.54
1.58
1.69
Resolution
9.26
8.64
9.03
8.64
9.02
8.64
7.6
8.51
8.93
8.93
8.64
1.58
1.61
1.59
1.56
8.64
8.97
8.98
9
6615
6562
6547
6341
Table 8: Results of amolodipine and metaprolol in marketed product
Marketed formulation
Brand-1
Brand-2
Brand-1
Brand-2
Drug
Amolodipine -5 mg
Amolodipine -5 mg
Metaprolol-25 mg
Metaprolol-25 mg
% Amount obtained
98.79± 0.86
99.59 ±0.90
99.06 ± 0.56
99.24 ±0.32
% RSD
0.30
0.32
0.56
0.48
Each value is a result of triplicate analysis.
Fig. 1: Chemical structures of amlodipine (A) RR and (B) S-isomers.
Fig. 2: Struture of Metaprolol
407
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Fig. 3: LOD Chromatogram of (R )-and (S )-Amolodpine and Metaprolol
Fig. 4: LOD Chromatogram of (R )-and (S )-Amolodpine and Metaprolol
Fig. 5(a): Linear calibration curve of (R)-Amolodipine besylate
408
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Fig. 5(b): Linear calibration curve of (S)-Amolodipine besylate
Fig. 5(c): Linear calibration curve of Metaprolol succinate
Fig. 6: Blank Chromatogram
409
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
Fig. 7: (R)- and (S )-Amolodipine Chromatogram
Fig. 8: Metaprolol Chromatogram
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
The United states pharmacopeia convention Revision Bulletin
official February 1, 2011.
www.drugleads.com/cds/amlodipine.html.
Chirally pure Antihypertensive drug (S)-Amolodipine-NCl
team: Dr. M.K. Gurjar, Dr. R. A. Joshi and Mrs. R. R. Joshi
www.drugleads.com/cds/metaprolol.html
Khopade S A, Jain. N.K. Difference Spectrophotometric
determination of amlodipine besylate. Indian drugs. 37; 2000:351.
Gohil K, Trivedi P and Molvi KI, Spectrophotometric analysis of
amlodipine besylate in bulk and in tablet dosage forms.Indian J
Pharma Sci. 2005; 67: 376-8
Ragno G, Garofalo A and Vetuschi C. Photodegradation
monitering of amlodipine by derivative spectrophotometry. J
Pharm Biomed Anal. 2002; 27: 19-24.
Chandrashekhar T G, Rao P S N, Smrita K.Analysis of
amlodipine besylate by HPTLC with fluorimetric detection: a
sensitive method for assay of tablets. J. Planar. Chromatogr.
Mod. TLC. 7; 1994: 458.
9. Pandya K K, Satia M, Gandhi T P. Detection and determination
of total amlodipine by high performance thin layer
chromatography:A useful technique for pharmacokinetic
studies. J. Chromatogr. B; Biomed. Appl. 667; 1995: 315.
10. Argekar A P, Powar S G. Simultaneous determination of
atenolol and amlodipine in tablets by high-performancethinlayer chromatography. J. Pharm. Biomed. Anal.21; 2000: 1137–
1142.
11. Ilango K, Kumar P B, Prasad V RV. Simple and rapid high
performance thin layer chromatographic determination of
amlodipine in pharmaceutical dosage forms. Indian J. Pharm.
Sci. 59; 1997: 336.
12. Meyyanathan SN and Suresh B. HPTLC method for
simultaneous determination of amlodipine and benazepril in
their formulations. J Chromatogr Sci. 2005; 43: 73-5.
410
Rao et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 5, 401-411
13. Halker U P, Bhandari N P, Rane S H. High
performanceliquidchromatographic
simultaneous
determination of amlodipine and enalapril maleate from
pharmaceutical preparation. Indian Drugs. 35; 1988: 168
14. Yeung P K F, Mosher S J, Pollack PT. High performance liquid
chromatography assay for amlodipine: chemical stability and
pharmacokinetics in rabbits.J.Pharm. Biomed. Anal. 9; 1991:
565.
15. Patki R V, Tamhanker C P, Tipnis H P Simple and rapid high
performance liquid chromatographic estimation of amlodipine
in pharmaceutical dosage forms. Indian Drugs 31; 1994: 560.
16. ShangF, ShangK. Determination of amlodipine in tablets
byHPLC. ZhoggnoYiyao Gangye Zazhi. 27; 1996:411.
17. Avadhanulu A B, Srinivas J S, Anjaneyulu Y.Reversed phase
HPLC determination of amlodipine in drugs and its
pharmaceutical dosage forms. Indian Drugs. 33; 1996: 36.
18. Sankar S R, Nanjan M J, Vasudevan M. Simultaneous estimation
of atenolol and amlodipinein formulations by reversed phaseHPLC. Indian J. Pharm. Sci. 59;1997: 171
19. Dhorda V J, Shetkar N B. Reversed phase liquid
chromatographic determination of ramipril and amlodipine in
tablets. Indian Drugs. 36; 1999: 638.
20. Naidu KR, Kale UN and Shingare MS. Stability indicating RPHPLC method for simulatneus determination of amlodipine and
benzapril hydrochloride from their combination drug product.
J Pharm Biomed Anal. 2005; 39: 147-55.
21. Shimooka K, Sawada Y, Tatematsu H.Analysis of amlodipine by
a sensitive high performance liquid chromatography method
withamperometricdetection. J.Pharm. Biomed. Anal. 7; 1989:
1267.
22. Josefsson M, Zackrisson A L, Norlander B.Sensitive
highperformance liquid chromatographic analysis of
amlodipine in human plasma with amperometric detection and
a single step solid phasesample preparation. J.Chromatogr.B;
Biomed. Appl. 672; 1995: 310
23. Bhatt J, Singh S, Subbaiah G, Shah B, Kambli S and Ameta S. A
rapid and sensitive liquid chromatography-tandem mass
spectrometry (LC-MS/MS) method for the estimation of
amlodipine in human plasma. J Biomed Chromatogr SciAppl.
2007; 21: 169-751998: 118.
24. Feng Y, Meng Q, Guo X. Human plasma amlodipine GC–MS
determination, Guandong Yaoxueyuan Xuebao.14 (111).
25. Veeraskaran V, Katakdhond SJ, Kadam SS and Jadhvi RR.
Simultaneus
spectrophatometric
estimation
of
hydrochlorthiazide and metoprolol tartarate from combined
dosage form. Indian Drug. 2001; 3: 187.
26. Pai PN, Shenoy KR and Pandey J. Simultaneus reverse phase
liquid chromatographic determination of metoprolol tartarate
and hydrochlorthiazide in tablets., Indian J Pharma Sci. 2005;
67: 608-10.
27. Marino EL, Modamio P, Lastra CF. et al. Development and
validation of liquid chromatography methods for the
quantitation of propranolol, metoprolol, atenolol, and
bisoprolol: application in solution stability studies. Int J Pharm.
130;1996: 137-140.
28. Aqil M, Ali A, Ahad A. et al. A validated HPLC method for
estimation of metoprolol in human plasma. Acta
Chromatographica. 19;2007: 130-140.
29. Leslie J, Mistry B, Eddington NE. A sensitive assay of metoprolol
and its major metabolite alpha hydroxyl metoprolol in human
plasma and determination of dextromethorphan and its
metabolite dextrophan in urine with high performance liquid
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
chromatographyand fluorometric detection. J Pharma Biomed
Anal. 16; 1998: 1041-1049.
Laer S, Albers S, Elshoff J. et al. HPLC quantification of
metoprolol with solid-phase Extraction for the drug monitoring
of pediatric patients. Biomed Chromatogr. 19; 2005:202-207.
Quarterman CP, Kendall MJ, Jack DB.Determination of
metoprolol metabolites in plasma and urine by electroncapture
gasliquidchromatography.
Journal
of
ChromatographyB:Biomedicalsciences and applications. 183;
1980: 92-98.
Lennard MS, Silas JH. Rapid determination of metoprolol and _
hydroxyl metoprolol in human plasmaandurinebyhigh
perfomance
liquid
chromatography.
Journal
of
Chromatography: Biomedical applications.272; 1983: 205-209
Pautler DB. Jusko WJ. Determination of metoprolol and
hydroxylmetoprolol in plasma by high performance liquid
chromatography.
Journal
of
Chromatography:
Biomedicalapplications.228; 1982: 215-222.
BalmerK.et.al. Determination of two metoprolol and two major
metabolites in plasma and urine by liquid chromatography and
flurometric detection. J Chromat. 417;1987: 357-365.
Godbillon J. et al. Determination of two metoprolol metabolites
in human urine by HPLC. J Chromatogr. 309; 1984: 198-202
Cooper SF, Li F, Cote M. Determination of the enantiomers of
metoprolol and its major metabolite in human urine by highperformance liquid chromatography with fluroscence
detection. J Chromatogr B. 668;1995: 67-75.
Chiu FC, Damani LA, Li RC and Tomlinson B. Efficient highperformance liquid chromatographic assay for the
simultaneous determination of metoprolol and two main
metabolites in human urine by solid-phase extraction and
fluorescence detection. J Chromatogr B Biomed Sci Appl. 1997;
696: 69-74.
Gyllenhaal O. et al Ion pair supercritical fluid chromatography
of metoprolol and related amino alcohols on dial silica. J
Chromatogr.1134; 2006: 305-310
Maria PQ, Anna MB, Luciano M, Guiseppe F and Fabio T.
Simultaneous determination of propranolol or metoprolol in
the presence of butyrophenones in human plasma by gas
chromatography with mass spectrometry. J Pharm Sci. 1993;
82:187-90.
Enantiomeric Separation and Determination of Stereospecific
DrugRelease from Marketed Racemic Amlodipine Besylate
Tablets by HPLC JaganMohan Somagoni, Sunil Reddy,
Someshwar Koorelli, Sarangapani Manda and Madhusudan Rao
Yamsani* Somagoni et al. Pharm Anal Acta 2011, 2:5.
Rapid enantioseparation of amlodipine by highly sulfated
cyclodextrins
using
short-end
injection
capillary
electrophoresis Zandkarimi M, * Shafaati A, Foroutan S.M,
3Charles A. Lucy. DARU Vol. 17, No. 4 2009.
Dongre VG, Shah SB, Karmuse PP. et al. Simultaneous
determination
of
metoprolol
succinate
and
amlodipinebesylatein pharmaceutical dosage form by HPLC. J
Pharma Biomed Anal. 46; 2008: 583-586
RP-HPLC Method of simultaneous estimation of amlodpine and
metaprolol
in
combined
dosage
form.
IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-9/NOV/010 CH.M.M.P
rasada Rao, S.A.Rahaman, Y.Ragjendra Prasad, P. Gangi Reddy.
Uv-Spectrophotometric Method For Simultaneous Estimation
Of Metoprolol And Amlodipine In Bulk And Their Formulation
Satyanarayana Rath. et al. / International Journal of Biological
& Pharmaceutical Research. 2011; 2(2): 50-54.
411